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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 610

Supplemental Assay Method for Potency Assay of Leptospira interrogans

Serogroup Icterohaemorrhagiae Bacterins

Date: December 23, 2022

Number: SAM 610.07

Supersedes: SAM 610.06, November 26, 2019

Standard Requirement: 9 CFR, part 113.102

Contact: Email: Methodsrequest.notification@usda.gov

Phone: Center for Veterinary Biologics, 515-337-6100

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product 

by USDA and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for Potency Assay of Leptospira interrogans

Serogroup Icterohaemorrhagiae Bacterins

Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

2.3 Animals

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents

3.4 Preparation of the sample

4. Performance of the Test

4.1 Vaccination of hamsters

4.2 Challenge procedure

4.3 Challenge of test hamsters

4.4 Observation of hamsters after challenge

5. Interpretation of the Test Results

6. Report of Test Results

7. References 

8. Summary of Revisions
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1. Introduction

This Supplemental Assay Method (SAM) describes the hamster vaccination-challenge method 

used to determine potency of Leptospira icterohaemorrhagiae bacterins as prescribed by title 9, 

Code of Federal Regulations (9 CFR), part 113.102.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Microscope with darkfield capability

2.1.2 Forceps, 5 1/2-inch, rat-tooth

2.1.3 Dissecting pins, 1- to 1 1/2-inch

2.1.4 Necropsy board

2.1.5 Stomacher® blender and sterile bags (alternatively, tissue grinders, 15 mL, 

TenBroeck may be used)

2.1.6 Balance, analytical

2.1.7 Calibrated weight set

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted as necessary.

2.2.1 Syringes, 1-mL tuberculin for animal injections and 3-mL for preparing 

challenge dilutions in 1% BSA glass serum vials.

2.2.2 Needles of appropriate gauge (e.g. 23g or 25g for hamster injections and 

larger 18g for preparing challenge dilutions in 1% BSA glass serum vials)

2.2.3 Scalpels

2.2.4 Glass screw-top tubes, 20 x 150-mm (or equivalent container)

2.2.5 Serum bottles with rubber stoppers (or equivalent container for preparing 

dilutions of liver homogenate)
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2.2.6 70% (v/v) ethanol

2.2.7 Microscope slides and coverslips

2.2.8 Pipettes, assorted sizes, cotton-plugged 

2.2.9 1% Bovine Serum Albumin Diluent (BSAD) National Centers for Animal 

Health (NCAH) Media #20133 in 10 mL glass serum vials and also in 20mm x 

125mm sc test tubes.

2.2.10 Sterile Petri plates 15mm X 100mm

2.2.11 0.85% NaCl solution (saline) NCAH Media #10191

2.2.12 L. icterohaemorrhagiae challenge culture IRP 655, frozen, hamstervirulent

2.3 Animals

2.3.1 Hamsters, adult, 50-90 g. Ten hamsters are required for each bacterin 

tested. Ten hamsters are always required per test session for nonvaccinated 

controls, and an additional twenty hamsters are required for LD50 titration if the 

back-titration exemption is not used (see CVB Public Notice 15-13). Sufficient 

hamsters should always be available to perform at least three in vivo serial 

passages of the Leptospira prior to challenge.

2.3.2 The hamsters must be obtained from the same source and colony. Use 

either all male or all female hamsters for any one test. 

2.3.3 House and feed all hamsters in an identical manner.

3. Preparation for the Test

3.1 Personnel qualifications/training

Technical personnel need a working knowledge of the use of general laboratory 

chemicals, equipment, and glassware and must have specific training and experience in 

the safe handling of live Leptospira spp. Personnel need specific training in the care and 

handling of laboratory hamsters and in the performance of this assay.

3.2 Preparation of equipment/instrumentation

3.2.1 Operate and maintain all equipment according to manufacturers’ 

recommendations and applicable in-house standard operating procedures.

3.2.2 Sterilize all glassware before use.
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3.2.3 Use only sterile supplies (pipettes, syringes, needles, etc.).

3.3 Preparation of reagents

3.3.1 Phosphate Buffer 25X Solution – NCAH Media #40105

Sodium Phosphate Dibasic Anhydrous 16.6 g

Potassium Phosphate Monobasic 2.172 g

Deionized water q.s. 1000 mL

Mix until dissolved. Filter sterilize (0.2-μm filter). Store refrigerated up to

6 months.

3.3.2 1% Bovine Serum Albumin Diluent – NCAH Media #20133

Phosphate Buffer 25X Solution (NCAH) Media #40105 40mL

Bovine serum albumin 10 g

Deionized water q.s. 960mL

Mix until dissolved. If necessary, adjust pH to 7.5 ± 0.1. Filter sterilize

(0.22-μm filter). Store at 20°- 25°C for no longer than 1 year.

 

3.3.3 0.85% saline – NCAH Media #10191

Sodium chloride 8.5 g

Deionized water q.s. 1.0 L

Autoclave at 121°C for 20 minutes. Store at 20°- 25°C for no longer than

1 year.

3.4 Preparation of the sample

3.4.1 Shake each bacterin to mix contents thoroughly.

3.4.2 Disinfect the top of the bacterin container with 70% ethanol.

3.4.3 Dilute each bacterin with saline so that 1 hamster dose (0.25 mL) is 

equivalent to 1/80 of the recommended host-animal dose.

Examples:

1. For 1-mL- host dose products, dilute the bacterin 1:20 in saline.

2. For 2-mL-dose products, dilute the bacterin 1:10 in saline. 

3. For 3-mL-host dose products, dilute the bacterin 1:6.7 (6.67) in saline.

4. For 4-mL-host dose products, dilute the bacterin 1:5 in saline.

5. For 5-mL-dose products, dilute the bacterin 1:4 in saline. 
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4. Performance of the Test

4.1 Vaccination of hamsters

4.1.1 For each bacterin to be tested, vaccinate 10 hamsters with 0.25 mL of 

appropriately diluted bacterin (see Section 3.4.3) using the route recommended by 

the manufacturer. If the recommended vaccination route is intramuscular, or if the 

product is labeled for either intramuscular or subcutaneous use, vaccinate the 

hamsters intramuscularly in the hind leg. If the label limits administration of that 

product to the subcutaneous route, vaccinate the hamsters subcutaneously in the

abdominal area. For all vaccinations, use a 1.0-mL syringe fitted with an 

appropriate size needle. 

4.1.2 Retain 10 nonvaccinated hamsters as controls. 

4.1.3 Retain 20 nonvaccinated hamsters to determine the LD50 of the challenge 

inoculum if required.

4.1.4 Cryopreserved challenge culture should be inoculated into the first 

hamsters for serial passage on the day of vaccination or within sufficient time to 

perform at least three serial passages prior to use in a regulatory test. Leptospira

passage through hamsters is conducted according to the current version of 

CVB-WI-0253, Leptospira Passage through Hamsters.

4.1.5 Challenge all hamsters with a virulent suspension of L. 

icterohaemorrhagiae 14 to 18 days after vaccination.

4.2 Challenge procedure

The challenge inoculum is a liver homogenate from a clinically ill hamster. The Center 

for Veterinary Biologics (CVB) maintains virulent, cryopreserved challenge organisms.

4.2.1 Select a clinically ill (preferably moribund) hamster from a group of 

hamsters that were infected 3 to 4 days previously with L. icterohaemorrhagiae.

4.2.2 Euthanize the hamster with CO2. Follow the euthanasia procedure 

approved by the Animal Care and Use Committee.

4.2.3 Pin the dead hamster to a posting board (ventral aspect up) and disinfect 

the skin with 70% ethanol.

4.2.4 Using aseptic technique, reflect the abdominal skin. Discard the 

instruments used to expose the abdominal musculature. Using fresh instruments, 

reflect the abdominal musculature to expose the abdominal viscera. Discard the 

instruments used to open the abdominal cavity.
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4.2.5 Using fresh instruments, aseptically remove approximately 1 gram of liver 

tissue. Measure the liver on an analytical scale in a tared sterile container in order 

to obtain 1.0 ± 0.1 gram of infected tissue. Aseptically place liver in a sterile 

blender bag. Add 9 mL of sterile BSAD to the bag. Thoroughly homogenize the 

liver with a Stomacher or tissue grinder, taking care to avoid foam formation. 

This suspension is considered the 1:10 dilution.

4.2.6 Prepare 5 additional serial tenfold dilutions (10-2 through 10-6) of the 

tissue suspension in BSAD (1.0 mL suspension + 9.0 mL diluent). Hold the 

dilutions at room temperature (20°- 25°C) and complete challenge inoculations 

within 1 hour after preparation of dilutions.

4.2.7 Place 2 drops of the 10-4 dilution on a microscope slide, cover with a 

coverslip, and examine under a 200X magnification with a darkfield microscope. 

The 10-4 dilution should have approximately 4 to 20 organisms per field.

4.2.8 If the 10-4 dilution has 4 to 20 organisms per field, then the 10-6 dilution

will be the challenge inoculum. (The challenge inoculum is the dose sufficient to 

deliver the required challenge of 10-10,000 LD50.) The challenge inoculum should 

be prepared in an appropriate sterile container.

Note: If the 10-4 dilution does not contain the appropriate number of 

spirochetes per field, determine which dilution contains between 4–20 

spirochetes/microscopic field. Then utilize the chart below to determine 

appropriate dilution to use as the challenge inoculum. 

Dilution Containing Challenge

4 - 20 spirochetes/field Inoculum Dilution

10-4 10-6

10-5 10-7

10-6 10-8

10-7 10-9

If the 10-4 dilution contains < 4 organisms per field, select another clinically 

ill hamster (Section 4.2.1) and prepare another challenge inoculum.

4.2.9 If the LD50 is required, prepare 4 additional tenfold dilutions beyond the 

dilution selected for the challenge inoculum. Retain these dilutions to determine 

the LD50 of the challenge inoculum. 

4.2.10 The remaining liver homogenate from the challenge inoculum may be 

used to infect additional hamsters to serve as a source of inoculum for future 
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potency tests. The dilution and dose volume should be tailored to suit the 

frequency with which serial passage is performed.

4.3 Challenge of test hamsters

4.3.1 Within 1 hour after preparation, inject intraperitoneally (IP) 0.2 mL of the 

challenge inoculum selected in Section 4.2.8 into each of the vaccinated hamsters 

and 10 nonvaccinated control hamsters. Use a 1.0-mL syringe fitted with an 

appropriate needle.

4.3.2 Inject 5 hamsters (0.2 mL, IP) with each of the dilutions prepared in 

Section 4.2.9 if a back-titration is required. These 4 groups of hamsters will be 

used to calculate the LD50 of the challenge inoculum. 

4.3.3 The 10-2 or -3 dilution should be retained and later tested for serogroup 

specificity through the current version of CVB-TWS-0048, Leptospira ELISA 

Screen.

4.3.4 Disinfect all work surfaces with 70% ethanol. Sterilize all contaminated 

equipment and supplies in the autoclave. 

4.4 Observation of hamsters after challenge

4.4.1 Observe all hamsters daily for 14 days following challenge. Record 

deaths.

Note: Moribund animals exhibiting clinical signs consistent with the expected 

disease pathogenesis that are unable to rise or move under their own power 

may be humanely euthanized and considered as deaths as outlined in 9 CFR 

117.4

4.4.2 At the end of the 14-day observation period, count the remaining hamsters 

and record results. 

4.4.3 Calculate the LD50 of the challenge inoculum using the Reed-Muench, 

Dragstedt-Behrens, or Spearman-Kärber method of calculation. The CVB 

typically uses the Dragstedt-Behrens method.

5. Interpretation of the Test Results

5.1 Interpret the results as described in 9 CFR 113.102.

5.2 The test is valid if 8 or more of the nonvaccinated hamsters that received the 

challenge inoculum are counted as deaths. An LD50 of 10-10,000 is an additional validity 

requirement without a backtitration exemption.
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5.3 If 3 or 4 vaccinates die in the first stage test, conduct a second stage test in a 

manner identical to the first stage. If the second stage is used, evaluate each serial 

according to the second part of the table. Serials pass or fail on the basis of cumulative 

results. Evaluate the results according to the following table:

Stage

Number of

Vaccinates

Cumulative

Number of

Vaccinates

Cumulative

Total Dead

Hamsters for

Satisfactory

Serial

Cumulative

Total Dead

Hamsters for 

Unsatisfactory Serial

1 10 10 2 or less 5 or more

2 10 20 5 or less 6 or more

6. Report of Test Results

Report the results of the test(s) as described by standard operating procedures.

7. References

Title 9, Code of Federal Regulations, part 113.102, U.S. Government Printing Office, 

Washington, DC.

CVB-WI-0253 Leptospira Passage through Hamsters.

CVB Public Notice 15-13 Option to Remove Back-titration Hamsters from In Vivo Potency 

Tests for Leptospira Serogroups Canicola and Icterohaemorrhagiae

8. Summary of Revisions

Version .07

• Formatting updated, updated coversheet, minor process updates

• 2.1.7 Added calibrated weight set

• 2.2.1, 2.2.2, 2.2.9, 2.2.11 Items were revised and updated

• 2.2 Updated to allow equivalent reagents/supplies to be used as necessary
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• 2.2.10 Removed P80-BA semi-solid medium and added Sterile Petri plates 15mm X 

100mm

• 2.2.12 Revised to L. icterohaemorrhagiae challenge culture IRP-655, frozen, hamstervirulent

• 2.3 Added reference to CVB Public Notice 15-13

• 3.3.1, 3.3.2, 3.3.3 Items were revised and updated. P80-BA semi-solid medium removed 

Phosphate Buffer 25X Solution was added

• 3.4.3 Revised with more dilution examples added

• 4.3.3 Changed recommended dilution from 10-4 to 10-2 or -3

• 4.4.1 Updated humane endpoints description for consistency

Version .06

• Formatting has been updated according to current practices.

• The LD50 calculation was clarified.

• The use of back-titration hamsters has been updated in agreement with current public 

guidance.

• Serogroup specificity testing and retention of shipping cultures has been updated based 

on current practices.

Version .05

• The Contact information has been updated, and minor edits to accurately reflect current 

practices.

Version .04

• The Contact information has been updated.

• The title has been changed to be more in agreement with current nomenclature 

practices.

• References to NVSL have been changed to NCAH throughout the document.

• Minor revisions have been made to clarify practices currently in use at the Center for 

Veterinary Biologics.
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Version .03

• Minor changes have been made to update the document to current formatting 

practices.

Version .02

This document was revised to clarify practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact 

the outcome of the test, the following changes were made to the document:

• 2.1 A Stomacher® blender and analytical balance have been added to the list of 

equipment and instrumentation.

• 2.2 The list of reagents and supplies has been updated to more accurately reflect what is 

used for this assay method.

• 3.3 The formulas have been updated to reflect that the solution may be stored at room 

temperature for up to one year.

• 3.3.3 P80-BA semi-solid medium has been added to the Preparation of Reagents section.

• References to internal CVB documents have been replaced with summary information.

• The contact person has been changed to Mary C. Rasmusson.